The current paradigm that Akkermansia muciniphila is a beneficial member of the human gut microbiome is based on an incomplete understanding of the physiological diversity and mechanistic activity across the lineage, as all previous work has focused on one described strain. The long-term goal is to help develop targeted, therapeutic uses of Akkermansia, either through stimulating endogenous strains with prebiotics or by administering specific strains as probiotics. The overall objectives for this application are to characterize the molecular mechanisms and immunogenic properties of genomically diverse strains of human-associated Akkermansia grown (i) on human milk oligosaccharides (HMO) and (ii) in the presence of bile. The central hypothesis of this work is that human-associated Akkermansia have evolved different growth efficiencies on HMO and bile stress responses that shape their immunogenic potential in a strain dependent manner. The rationale for this work is that if we are to use Akkermansia for the therapeutic treatment of metabolic disorders or other gastrointestinal diseases, then we need to design biologically informed treatment strategies that promote or introduce select strains for optimal health outcomes. The central hypothesis will be tested by pursuing two specific aims: 1) Identify the molecular mechanisms and immunogenic properties of Akkermansia grown on HMO; 2) Identify the bile resistance mechanisms and immunogenic properties of Akkermansia grown in the presence of bile. Under the first aim, targeted gene expression studies coupled with cloning and functional assays will be used to identify glycoside hydrolase enzymes involved in growth on HMO in three genomically diverse Akkermansia isolates. Concurrently, targeted metabolomics analyses will be used to quantify HMO consumption and fermentation end products. Lastly, co-culture experiments with HMO grown Akkermansia cells and human epithelial cells will be used to measure bacterial binding efficiency and the immune response of the epithelial cells. For the second aim, transcriptomic and proteomic profiling, coupled with targeted metabolomics (i.e. bile acid composition and microbial extracellular polysaccharides) will be used to characterize the bile stress response of the three Akkermansia isolates. Similar to aim 1, co- culture experiments with bile grown Akkermansia strains and human epithelial cells be used to measure bacterial binding efficiency and the host immune response. The research proposed in this application is innovative because it represents a substantive departure from the status quo by providing insights into the physiological diversity and molecular mechanisms across the Akkermansia lineage. The proposed research is significant because it will help define the physiological landscape across the lineage, thereby opening new horizons for biologically informed treatment strategies that promote or introduce select Akkermansia strains for optimal health outcomes.